In the central nervous system, most excitatory synapses terminate on dendritic spines, small postsynaptic compartments emanating from the dendritic surface. Ca2+ influx into spines activates a signaling network required for diverse forms of synaptic plasticity. In particular, the family of ~150 small GTPase proteins is important for many aspects of synaptic plasticity, including regulation of the actin cytoskeleton, membrane trafficking, vesicular transport and gene transcription. In this study, we will develop a technique to monitor the activity of more than 60 small GTPase proteins in single dendritic spines in brain slices. To do so, we will develop scalable designs and optimization schemes to make fluorescence resonance energy transfer (FRET)-based sensors reporting small GTPase activity with high sensitivity. To quantitatively image FRET signal with high sensitivity and resolution in light scattering brain tissue, we will use 2-photon fluorescence lifetime imaging microscopy (2pFLIM). Our preliminary data demonstrates that our design can be applied to many small GTPase proteins. Using these sensors, we will screen small GTPase proteins activated by NMDA receptors, and image their activity in single dendritic spines undergoing structural and functional plasticity. Our specific aims are 1) to develop and test sensors for small GTPase proteins, 2) to screen small GTPase proteins for those activated by Ca2+ through NMDA receptors, 3) to measure the spatiotemporal dynamics of selected small GTPase proteins in single dendritic spines. This study will provide insights into how the activity of small GTPase proteins is coordinated in spines to produce structural and functional plasticity of dendritic spines, and will illuminate the molecular mechanisms of synaptic plasticity and ultimately learning and memory.